Project Summary: Eukaryotic cells partition ribosomes and mRNAs between the cytosol and endoplasmic reticulum (ER) as a mechanism for compartmentalizing secretory and membrane protein synthesis to the ER. In contrast to prominently studied examples of RNA localization, where localized RNAs traffic to specialized regions of the cell periphery as translationally-silenced ribonucleoprotein particles (RNPs) and undergo translation once anchored at their distal destination, RNA localization to the ER occurs via the translation-dependent signal recognition particle (SRP) pathway. In the SRP pathway, all mRNAs initiate translation in the cytosol, with signal sequence-encoding mRNAs undergoing co-translational trafficking to the endoplasmic reticulum (ER). Upon termination, ER-bound ribosomes return to the cytosol, thereby completing a cycle of selective mRNA localization and ribosome exchange. With the benefit of robust in vitro biochemical assays and atomic structures of pathway components, the mechanism of SRP selection is well understood. The question of SRP pathway function in vivo has, however, remained largely unexplored. Two sets of observations suggest that the in vivo mechanism of mRNA localization to the ER is divergent from current models. One, analyses of the mRNA compositions of cytosolic and ER-bound ribosomes in mammalian tissues and cells, yeast, and fly demonstrate that cytosolic protein- encoding mRNAs are broadly represented on the ER, though they lack encoded signal sequences. Two, genetic inactivation of SRP or SRP receptor in yeast is tolerated, as is stable knockdown of SRP and SRP receptor expression in mammalian cells, findings which suggest a selective, rather then general, role for the SRP pathway in RNA localization to the ER. These observations point to significant gaps in our understanding the mechanisms of translational compartmentalization in vivo. Here we propose three aims to investigate this fundamental question. In the first aim, we will generate SRP, SRP receptor-? and SRP receptor-? CRISPR- Cas9 knock-out and inducible shRNA knock-down human cell lines, as model systems to study translational compartmentalization. In a second aim, RNA-Seq/Ribo-Seq studies of parental and SRP pathway-mutant cell lines will be performed to define SRP, SRP receptor-?, and SRP receptor-? function in mRNA and ribosome localization to the ER, and to identify candidate mechanisms that compensate for SRP pathway inactivation. In a third aim, we will use 4-thiouridine pulse-labeling, SRP pathway mutant cell lines, and pharmacological translation inhibitors to define the subcellular site(s) of translation initiation and the role of the SRP pathway in directing the pioneer localization step pathway for newly exported mRNAs. These studies are expected to provide critical new insights into mechanisms of mRNA and ribosome compartmentalization in eukaryotic cells and thereby accelerate our understanding of translational regulation in homeostasis and disease.